Title of Invention

A PROCESS FOR THE PREPARATION OF ADHESIVE EXOPOLYSACCHARIDE BY BACILLUS SP.

Abstract A process for the preparation of adhesive exopolysaccharide by Bacillus spp which comprises cultivating a polysaccharide producing strain of Bacillus spp NIO-FB-007 having characteristics as herein described in known nutrient medium supplemented with monosaccharide or disaccharide as carbon source for about 4-5 days at a temperature ranging 28-30°C and at pH ranging 7-10, separating supernatant from the culture broth by centrifugation at about 8000 rpm, concentrating the cell free supernatant using conventional technique, recovering and purifying the adhesive exopolysaccharide by conventional precipitation and dialysis methods
Full Text This invention relates to a process for the preparation of adhesive polysaccharide. More particularly, this invention relates to a microbial process whereby a extracellular microbial polysaccharide also known as exopolysaccharide possessing useful adhesive properties can be produced by means of polysaccharide producing strain of microorganism.
The interest in microorganisms as producers of high molecular- weight polysaccharides has greatly increased in recent years, since these biopolymers often show advantages over the polysaccharides currently in use, which are mostly extracted from plants or marine macroalgae ( Colwell RR, Pariser E R & Sinskey A J, 1985, Biotechnology of Marine polysaccharides, Hemisphere Publishing House, Washington).
Exoploysaccharides have many applications in industry. They are used as stabilizers, emulsifiers and thickners in food industry. In oil industry they are used for both drilling and oil recovery operations. Exopolysaccharides are also used in the manufacture of paper and textiles. Recently efforts are on to assess their use as anti - cancer compounds. Many types of microorganisms are known to produce exopolysaccharides. They are also used as adhesives. Microbial exopolysaccharides can possess unique properties and can be produced to uniform specification.
We have discovered a polysaccharide possessing adhesive properties which are similar to those of commercial adhesives, suggesting commercial application. This

Polysaccharide can be prepared by cultivating a polysaccharide producing strain of Bacillus sp. in nutrient medium.
The prior art process employed for the production of exopolysaccharides is a batch process. Using this prior art several novel exopolysaccharides have been isolated from marine bacteria. Reference may be made to U S patent No. 4298725, Process for the preparation of polysaccharide, issued on November 3, 1981 to Williams A G, Lawson C J, Wimpenny J W T; Christensen B E, Kjosbakken J, Smidsrod, O 1985; Partial chemical and physical characterization of two extracellular polysaccharides produced by marine periphytic Pseudomonas sp strain NCMB 2021. Appl. Environ. Microbiol. 50: 837-945; William et al., 1977. Exopolysaccharide production by Pseudomonas WCIB 11264 grown in batch culture. J. Gen. Micro., 102, 13-21. In the course of searches for new marine biopolymers of potential use to industry, we have discovered a exopolysaccharide possessing adhesive properties which are similar to those of commercial adhesives. The main object of the preset invention is to provide a process for the preparation of adhesive polysaccharide by means of polysaccharide producing strain of a bacterium designated as Bacillus sp. and deposited at National Institute of Oceanography culture collection facility bearing culture number NIO-AB-007.
Accordingly the present invention provides a process for the preparation of adhesive exopolysaccharide by Bacillus sp. which comprises cultivating a polysaccharide producing strain of Bacillus sp. NIO-FB-007 and deposited at National Institute of Oceanography having characteristics as herein described in known nutrient medium

supplemented with monosaccharide or disaccharide as carbon source for about 4-5 days at a temperature ranging 28-30°C and at pH ranging 7-10, separating supernatant from the culture broth by centrifugation at about 8000 rpm, concentrating the cell free supernatant using conventional technique, recovering and purifying the adhesive exopolysaccharide by conventional precipitation and dialysis methods as herein described.
In an embodiment of the present invention, Bacillus sp was isolated from the biofilm
developed on mild steel panels immersed in surface waters of the Dona Paula Bay, Its
morphology and physiology can be summarised as follows (all temperatures in degree
centigrades):
Morphology on ZoBell Marine Agar at 28+2°C
Gram - positive, motile rods, colonies: 1 - 2 mm, whitish, opaque, circular, entire,
smooth, soft, not easily dispersed .
Physiology at 28° C
Catalase +
Oxidase +
Hugh & Leifson Facultative
Methyl red _
Voges - Proskauer test +
Indole
Triple sugar Iron Alkaline Slants; Acid Butt
Peptone Water sugars
Fructose +
Xylose +
Glucose +
Sucrose +
Maltose +
Gelatin hydrolysis _
Urease _
Nitrate reductase +
_ = Negative; + = Positive

In another embodiment of the present invention the microorganism can be cultivated under aerobic conditions, in any convenient medium in which it will grow and produce extracellular polysaccharide. The typical medium include a chemically defined medium such as that described by Rodrigues and Bhosle (Biofouling, 4: 301 -308,1991) with supplementary carbon source of, for example, glucose or sucrose or xylose. A supplement of about 1 to 2 % W/V V is desirable.
In another embodiment the process for the preparation of adhesive polysaccharide which comprises cultivating a polysaccharide producing strain of Bacillus sp, having characteristics as herein described in known nutrient medium supplemented with disaccharide as carbon source, in a known manner at least for 5 days, separating supernatant from cells, then recovering and purifying by conventional precipitation and dialysis methods the extracellular adhesive polysaccharide from the cell free supernatant.
This invention particularly relates to a microbial process whereby a extracellular microbial poly saccharide also known as exopolysaccharide possessing useful adhesive properties can be produced by means of polysaccharide producing strain of microorganism designated as Bacillus sp and deposited at the NIO culture collection facility bearing number NIO- FB- 007 is used for the preparation of adhesive polysaccharide.

In another embodiment the nutrient medium has the following composition. Nutrient Medium Composition
NaCI 30.00 g
Kcl 0.75 g
MgSO4 7.00 g
NhUCI 1.00g
K2HP04 (10%) 7.00ml
KH2P04 (10%) 3.00ml
Trace metal solution 1.00ml
Distilled water 1000.00 ml
pH 7.5
In yet another embodiment the nutrient medium was supplemented with the trace metal solution and the said trace metal solution has the following composition.
Trace metal solution composition
H3B03 2.85 g
MnCI2.7 H20 1.80 g
FeS04.7H20 2.49 g
Na-Tartarate 1.77 g
CuCI2 0.03 g
ZnCI2 0.02 g
CoCI2 0.04 g
Na2Mo04.2H20 0.02 g
Distilled water 1000.00 ml
In yet another embodiment monosaccharides used are mannose, galactose, xylose, glucose and employed in the range of 0.5 to 5% W/V.

In another embodiment disaccharide used is sucrose and employed in the range of
0.5 to 5% W/V.
In another embodiment the cultivation is effected at temperature ranging between 28
to 30°C
In another embodiment the cultivation is effected at pH ranging between 7 to 10.
In yet another embodiment the polysaccharides are recovered by precipitation using
aliphatic alcohol such as methanol, ethanol and isopropanol.
In another embodiment the purification is effected by conventional methods of precipitation and dialysis.
In yet another embodiment the culturing of Bacillus sp,may be effected in a batch-wise manner, according to conventional practice. With a monosaccharide or disaccharide particularly sucrose - supplemented medium, the sucrose conversion is about 30%.
.In yet another embodiment polysaccharide production is found to be enhanced when an excess of carbon source is present, under nitrogen - limited conditions.
In yet another embodiment the extracellular polysaccharide may be isolated from the culture supernatant (free from cells) by precipitation with organic solvent such as aliphatic alcohol and deionized by using dialysis bags ( MW cut off of 8000 dalton).

After dialysis, the isolated material can be precipitated and dried at ~50°C in an oven to give the purified extracellular polysaccharide.
Chemical characterization using the Gas Chromatographic, Fourier Transformed Infra Red Spectrometer and Spectrophotometric methods have shown that the purified polysaccharide to be a polysaccharide having the following components: The polysaccharide is composed of xylose ( 53 %) and glucose (23 %) as major components while other monosaccharides such as arabinose, mannose and galactose were present in small amounts (~ 5 to 7 %).
The purified polysaccharide contains pyruvate ( -13.6%) as the most abundant non-sugar component. Sulphate, uronic acids, hexosamines and protein were also present in small quantities.
Purified polysaccharides was found to be very adhesive. Adhesiveness of the polysaccharide appears to be better than that of commercial fevicol and relatively less than that of commercial quickfix when tested on polyvinyl chloride and fiberglass surfaces. A typical shear stress required to break the adhesive bond between the polysaccharide and the surface such as metal and PVC is -10 Kg/cm2.
The following examples illustrates the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1: Growth and exopolysaccharide production by Bacillus sp*
Extracellular polysaccharide production by Bacillus sp was followed in a batch culture. The Bacillus sp culture was grown in a five litre flask containing two litre of growth medium as herein before described and supplemented with 1 % of sucrose as the carbon source for 5 days. Aeration was provided by shaking the culture flask at 150 RPM using the Rotary shaker. The logarithmic growth continued for 32 hours during this stage polysaccharides
H2SO4
production could be detected by the phenol -H2SO4technique. Although exopolysaccharides production commence during the first 4 hours of the exponential phase of growth, the production was highest during the stationary growth phase (>32 hours) Figure 1 represent production of adhisive polysaccharide by the bacterium Bacillus sp, NIO isolate number MCRD-FB-001. This production pattern is typical of a secondary metabolite.
Example 2: Isolation and partial purification of exopolysaccharide
The Bacillus culture was grown in 2 liter of defined growth medium as described above supplemented with 1 % of sucrose as carbon source for 5 days. Cells were removed by centrifugation at 8000 RPM and 4°C. Cell free supernatant was concentrated to 40 milliliters using the conventional ultrafilteration technique. Exopolysaccharide was isolated from the 40 milliliters of the concentrated cell free supernatant by adding 120 milliliters of ethyl alcohol. The precipitated

exopolysaccharide was collected using a glass rod and dissolved in 10 milliliters of
distilled water. Exopolysaccharide solution was transferred to dialysis bags
8000 MW cut off) and dialysed overnight against distilled water to obtain partially purified exopolysaccharide. The obtained 15 milliliters of dialysed exopolysaccharide solution was then treated with 45 milliliters of ethyl alcohol to precipitate the exopolysaccharide. The exopolysaccharide was collected by centrifugation at 8000 RPM and dried at 50°C.
Example 3: Effect of growth medium pH on exopolysaccharide production
Ten milliliters of the growth medium as defined above was transferred to 5 conical flasks of 100 milliliter capacity. The pH of the growth medium was adjusted to 5 to 10 using 1 N NaOH. The flasks were inoculated with 2% of 48 hour old culture of Bacillus sp. The flasks were incubated at room temperature ( 28 to 30 °C) for 5 days. Culture broth was centrifuged at 8000 RPM to remove the cells. Cell free supernatant was dialysed using dialysis bags ( 8000 MW cut off) against distilled water. One milliliter of dialysed supernatant solution was then used to estimate the content of exopolysaccharide in the solution using the conventional phenol -sulphuric acid technique.. The exopolysaccharide production increased with increase in the pH of the growth medium and highest amount of exopolysaccharide was obtained when the growth medium pH was 10 ( Table 1).
10

Example 4: Effect of NH4CI concentration on the exopolysaccharide production
Growth medium ( 10 milliliters) as defined above was transferred to 100 milliliters of flasks to which 0.005 to 0,1 % of NH4CI was added. The flasks were inoculated with 2% of 48 hour old culture and incubated at room temperature for 5 days. Cells were removed by centrifugation at 8000 RPM. Cell free supernatant (5 milliliters) was dialysed using dialysis bags. One milliliter dialysed solution was used to assess exopolysaccharide content using conventional phenol - sulphuric acid technique. As the ammonium chloride concentration increased the exopolysaccharide production also increased and highest exopolysaccharide production was observed when the ammonium chloride concentration was 0.06% (Table 2).
Example 5: Effect of phosphate concentration on exopolysaccharide production
Growth medium as defined above was supplemented with various concentration of phosphate ranging from 0.025 to 0.2 % and inoculated with 2% of 48 hour old culture of Bacillus sp, incubated at room temperature for 5 days. Cells were removed by centrifugation at 8000 RPM. Five miliiliters of the cell free supernatant was dialysed using a dialysis bag (8000 MW cut off) using distilled water. Exopolysaccharide content of dialysed cell free supernatant was assessed by the

phenol - sulphuric acid technique. Exopolysaccharide production increased with the increase in phosphate concentration and was highest when the phosphate concentration was 0.15 % (Table 3).
Example 6: Effect of calcium chloride on exopolysaccharide production
The effect of calcium chloride concentration was evaluated by adding various concentrations (0.005 to 0.05%) of calcium chloride to the growth medium defined as above. Growth medium was inoculated with 2% of 48 hour old culture of Bacillus sp and incubated at room temperature for 5 days. Cells were removed by centrifugation at 8000 RPM and cell free supernatant was dialysed using dialysis bags (8000 MW cut off). Exopolysaccharide concentration in dialysed cell free supernatant was assessed by phenol - sulphuric acid technique. Of these, 0.02% calcium chloride produced highest amount of the polysaccharide (Table 4).
Example 7: Effect of magnesium sulphate on exopolysaccharide production
Magnesium sulphate was added at various concentrations (0.1 to 0.9%) to the growth medium defined as above, inoculated with 2% of 48 hour old culture of Bacillus sp and incubated for 5 day at room temperature to assess its effect on the production of exopolysaccharide. Cells were removed by centrifugation at 8000 RPM. Cell free supernatant was dialysed using dialysis bags (8000 MW cut off). Exopolysaccharide content of the cell free supernatant was assessed by the phenol

- sulphuric acid technique. Of the various concentration used, 0.3% of magnesium sulphate produced highest amount of exopolysaccharide (Table 5).
Example 8: Effect of sugars on exopolysaccharide production
The effect of various sugars was assessed by supplementing the growth medium defined as above with 1 % of either galactose, xylose, sucrose, maltose and glucose. Growth medium was inoculated with 2% of 48 hour old culture of Bacillus sp and incubated at room temperature for 5 days. Cells were removed by centrifugation at 8000 RPM. Cell free supernatant was dialysed using dialysis bags. Exopolysaccharide content of the cell free dialysed supernatant was estimated by the phenol - sulphuric acid technique. Of the various sugars used, sucrose produced highest amount of exopolysaccharide (Table 6). Polysaccharide production generally increased when sucrose concentration increased from 1 to 5% (Table 7).
The conclusion of the present invention is that a polysaccharide possessing useful adhesive properties is prepared by cultivation of Bacillus sp. The polysaccharide possess adhesive properties which are similar or better than those of the some commercial products currently available in the market.
The main advantages of the present invention are: It is a naturally produced polysaccharide. It exhibit good adhesive properties for example, 10kg/cm2. It is biodegradable. It may find application in various industries

Table 1. Effect of pH on the production of Exopolysaccharides by Bacillus spec/es.
pH EPS

(Table Removed)
Table 2. Effect of Nitrogen concentration on the production of Exopolysaccharides
NH4CI2 EPS
Concentration (µg/rnl)
0.005 399.4
0.03 446.2
0.06 561.6
0.08 535.7
0.10 486.7
Table 3. Effect of Phosphate concentration on the production of Exopolysaccharides
(Table Removed)
Table 4. Effect of calcium chloride (CaCI2) concentration on the production of Exopolysaccharides by Bacillus Species.
CaCI2 EPS
Concentration (µg/ml)

(Table Removed)
Table 5. Effect of MgSO4 concentration on the production of Exopolysaccharides
byBacillusspecies.
MgS04 EPS
Concentration (µg/ml)
0.1 330.7
0.3 368.4
0.5 199.9
0.7 188.9
0.9 214.7
Tables. Effect of carbon sources on the Production of Exopolysaccharides by Bacillus species.
(Table Removed)

Table 7. Effect of sugar concentration on the production of Exopolysaccharides by Bacillus species.
Sucrose EPS
Concentration
(Table Removed)



We Claim:
1. A process for the preparation of adhesive exopolysaccharide by Bacillus sp.
which comprises cultivating a polysaccharide producing strain of Bacillus sp.
NIO-FB-007 and deposited at National Institute of Oceanography having
characteristics as herein described in known nutrient medium supplemented with
monosaccharide or disaccharide as carbon source for about 4-5 days at a
temperature ranging 28-30°C and at pH ranging 7-10, separating supernatant
from the culture broth by centrifugation at about 8000 rpm, concentrating the cell
free supernatant using conventional technique, recovering and purifying the
adhesive exopolysaccharide by conventional precipitation and dialysis methods as
herein described.
2. A process as claimed in claim 1, wherein the nutrient medium has the following
composition.
NaCI 30.00 g
KCI 0.75 g
MgS04 7.00 g
NH4CI 1-OOg
K2HP04 (10%) 7.00 ml
KH2P04 (10%) 3.00 ml
Trace metal solution 1 -00 ml
Distilled water 1000.00ml
pH 7.5

3. A process as claimed in claims 1 -2, wherein the nutrient medium supplemented
with the trace metal solution and the said trace metal solution has the following
composition.
H3B03 2.85 g
MnCI2.7H2O 1.80g
FeSO4.7 H2O 2.49 g
Na-Tartarate 1.77g
CuCI2 0.03 g
ZnCI2 0.02 g
CoCI2 0.04 g
Na2Mo04 2H20 0.02 g
Distilled water 1000.00 ml
4. A process as claimed in claim 1 , wherein Bacillus sp. NIO-FB -007 used is an
isolate from the biofilm of steel panels immersed in surface water of Dona Paula
Bay.
5. A process as claimed in claims 1 -3, wherein monosaccharides used are mannose,
galactose, xylose, glucose and employed in the range of 0.5 to 5% W/V of the
nutrient medium.

6. A process as claimed in claims 1-4, wherein disaccharide used is sucrose and
employed in the range of 0.5 to 5% W/V of the nutrient medium.
7. A process as claimed in claims 1-4, wherein the exopolysaccharides are
recovered by precipitation using aliphatic alcohol selected from methanol, ethanol
and isopropanol.
8. A process for the preparation of adhesive exopolysaccharide by Bacillus spp
substantially as herein described with reference to the examples accompanying
this specification.

Documents:

492-del-1999-abstract.pdf

492-del-1999-claims.pdf

492-del-1999-correspondence-others.pdf

492-del-1999-correspondence-po.pdf

492-del-1999-description (complete).pdf

492-del-1999-form-1.pdf

492-del-1999-form-19.pdf

492-del-1999-form-2.pdf


Patent Number 215482
Indian Patent Application Number 492/DEL/1999
PG Journal Number 11/2008
Publication Date 14-Mar-2008
Grant Date 27-Feb-2008
Date of Filing 31-Mar-1999
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001,INDIA
Inventors:
# Inventor's Name Inventor's Address
1 NARAYAN BABURAO BHOSLE NATIONAL INSTITUTE OF OCEANOGRAPHY, CSIR C/O NIO, DONA PAULA, GOA-403 004,INDIA
2 FRADDRY JOSE D'SOUZA NATIONAL INSTITUTE OF OCEANOGRAPHY, CSIR C/O NIO, DONA PAULA, GOA-403 004,INDIA
PCT International Classification Number A01N 63/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA